The expression, purification, and classification of gbr22 using E. coli Introduction
Protein expression and purification is a complicated procedure for those who without much experience since its successfulness is highly variant depending on which materials and methods are used. Although there are no absolute guides for determining which strategies are best, it is possible to suggest a starting point for projects based on experience and statistics. Here we will list many factors that can affect the outcome of the purification process.
The domain of interest must be carefully selected. Since the over-expression of the entire domain of the protein may not be needed, it is important to select the correct N and C terminus. Even a slight difference in the target selection can have a strong effect on the protein behavior. When selecting the domain, it is necessary to consider the protein class (e.g. globular, rod, integral, etc...). A valuable way to guard against errors in domain selection is by using a multiple sequence alignment. With BLAST, the sequences can be compared in order to see the "globularity". It is also important to select the appropriate host for the target gene. The most common choices are E. coli and yeast. E. coli is the cheapest and quickest way to obtain protein, but yeast has the advantage of producing much higher concentrations. An affinity tag should also be added to the protein since they aid in purification but have little effect on biological function. There are many choices of tags, although none are significantly better than the others. A small scale test can be useful for determining the final scale of the actual study since the conditions of the surroundings can affect the growth rates and protein output is unknown. The amount of protein that should be loaded into the column should also be slightly over what is the predicted amount. Proteins should be characterized after purification to ensure that only the target is present. This can be confirmed with an SDS-PAGE. The location and intensity of the bands can be used to determine if the purification was successful. The concentration of protein produced can be found using UV spectrophotometry.
In this lab, we over-expressed gbr22 in E. coli by cloning and harvested the pure protein. The results were finally confirmed with an SDS-PAGE. We can use the purification of gbr22 as a model for future protein generation in virtual drug screening. As we move from in silico to wet lab testing, we will need to test the activity of the target without other confounding chemicals. This process will allows us to obtain the proteins needed.
Materials and Methods
Protein Expression: The gene gbr22 was cloned and inserted into pGEM-gbr22. E. coli was then transformed by adding 25ul of bacteria to two 14 ml clear, sterile round-bottom tubes and adding 1ul of pGEM-gbr22 to one of the tubes("DNA tube"). After chilling both tubes on ice for 30 minutes, the two tube were heat shocked in a 42 oC water bath and placed back in ice for 2 more minutes. 200 ul of SOC media were added to the two tubes and incubated for 30 minutes at 37 oC at 250 rpm. Afterwards, 50ul of each bacteria tube were placed in two agar AMP plates and stored in a 37 oC incubator for at least 24 hours. Starter cultures were then grown by adding a colony to a LB/amp media and placing inside a 37 oC shaking incubator at 200-350 rpm for 8 hours. 25 ml of the "DNA LB" was then transferred to two 125 ml Erlenmeyer flasks. After 24 hours, the cell were harvested by pouring the bacteria into a 50ml conical tube and centrifuging for 10 minutes at 5000 rpm at 4 oC. The pellet from each centrifuged tube was saved after draining the spent media. 2.5 ml of 1x PBS and lysozyme was added until final concentration was 1mg/ml. The tube was then placed in a -20 oC freezer.
Protein Purification: The E. coli cells were lysed by adding 2 ul of Benzonase to each 50 ml conical. The lysate was then cleared by centrifuging for 20 minutes at 14000 rpm at 4 oC. The liquid supernatant was then extracted and placed in 15 ml conical tubes. The lysate was further filtered with a .22 um syringe filter. The lysate was finally purified using a hexahistone affinity tag and Ni-NTA resin. The purified protein was then run through a UV-Vis spectrophotometer at 280 nm and at maximal wavelength.
Protein Characterization: The purified protein was run through an SDS-PAGE. The MW standard used was PageRuler Prestained Protein Ladder. The gen was run for 25 minutes at 200 V.
Results
Figure 1A: The plate with ampicillin had several transformed, purple colonies of bacteria. The bacteria successfully took up the plasmid and began expressing the purple protein along with ampicillin resistance.
The pGEM-gbr22 had 10 colonies growing on it.
Figure 1B: The control plate with ampicillin
The control plate with ampicillin had no growth since the bacteria were not transformed by the plasmid. Therefore, the bacteria were not resistant to the antibiotic. The lack of growth indicates that the techniques used throughout the lab did not involve contamination with the other culture.
Figure 1C: The "fun plate" that we coughed on exhibited no bacterial growth.
Figure 2: BL21(DE3) pGEM-gbr22 suspended in LB-amp solution after 21 hours of incubation at 37 degrees Celcius.
The starter cultures had a purple/pink tint, indicating that protein was produced, but had impurities.
Figure3: Pellets of BL21(DE3) pGEM-gbr22 after centrifuging for 10 minutes at 5000 rpm and 4 degrees Celsius. The wet pellet weights were .45 and .49 grams.
The pellets, although filled with cell components became much more purple since they were more concentrated.
Figure 4: Elution 1 and 2 are shown. Elution 1 contains the protein gbr 22 as indicated by the purple color. The 2nd elution does not contain protein.
Figure 5A: Wet SDS-PAGE of samples 2-6 (lanes 5-10).
The SDS-PAGE gel showed decreasing amounts of protein in each lane 5-10. Sample 1 is missing since the tube could not be found.
Figure 5B: Dried SDS-PAGE gel.
Figure 6A: Trial 1 of spectrograph at 280nm.
Figure 6B: Trial 2 of spectrograph at 280nm.
Figure 7A: Trial 1 of UV-vis spectrograph at max wavelength.
Figure 7B: Trial 2 of UV-vis spectrograph at max wavelength.
The band in sample 5 (purified protein) was lined with the 25 kDa mark. There was a large amount of contaminate from the previous sample, indicating that the initial imidazole flow did not work. The estimated purity was 10% for elution 1. However, elution 2 had a very defined single line, even though there was not supposed to be one. The purity for sample 6 is close to 100%. This suggests that we used the incorrect imidazole solution when performing the elutions.
Discussion In this "pilot" lab for protein purification, there were many errors that may have contaminated the results. Although there were many major errors, it seems that we were still able to obtain a small amount of reasonably pure gbr22.
One particularly large source of error was when we were performing the elutions. When the lysate was placed inside the column, the tip was not capped and flowed into the waste beaker. We had to reuse this liquid and ran it through the column again, increasing the potential for contaminants. Also, the wrong imidazole concentration was used initially for elution 2. As seen in figure 5B, the elution 1 has many bands even though this is the purified protein and there should be one band. Elution 2, which is supposed to be devoid of any bands, shows one band. This indicates that the procedure was performed incorrectly or that the optimal imidazole concentration was actually 250mM. The spectrophotometer results also indicated a large amount of error. The calculated yields were off by more than a factor of 2. This suggests that there were other contaminants in the sample that contributed to the spectrograph.
Many of these errors could be attributed to clumsiness and inexperience handling the materials (especially adding 5 ladders to the gel). Our results could be improved by simply being more careful and taking more time completely the delicate parts of the procedure.
Conclusion
Despite various errors, we still were able to obtain a pure sample of gbr22 so we can still consider this lab to be successful. We can use this purification technique in the future to produce pure samples of a target protein for protein assay when testing ligands in virtual screening. In addition, purification can be applied on a larger scale to actually produce successful drugs.
References
Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
The expression, purification, and classification of gbr22 using E. coliIntroduction
Protein expression and purification is a complicated procedure for those who without much experience since its successfulness is highly variant depending on which materials and methods are used. Although there are no absolute guides for determining which strategies are best, it is possible to suggest a starting point for projects based on experience and statistics. Here we will list many factors that can affect the outcome of the purification process.
The domain of interest must be carefully selected. Since the over-expression of the entire domain of the protein may not be needed, it is important to select the correct N and C terminus. Even a slight difference in the target selection can have a strong effect on the protein behavior. When selecting the domain, it is necessary to consider the protein class (e.g. globular, rod, integral, etc...). A valuable way to guard against errors in domain selection is by using a multiple sequence alignment. With BLAST, the sequences can be compared in order to see the "globularity". It is also important to select the appropriate host for the target gene. The most common choices are E. coli and yeast. E. coli is the cheapest and quickest way to obtain protein, but yeast has the advantage of producing much higher concentrations. An affinity tag should also be added to the protein since they aid in purification but have little effect on biological function. There are many choices of tags, although none are significantly better than the others. A small scale test can be useful for determining the final scale of the actual study since the conditions of the surroundings can affect the growth rates and protein output is unknown. The amount of protein that should be loaded into the column should also be slightly over what is the predicted amount. Proteins should be characterized after purification to ensure that only the target is present. This can be confirmed with an SDS-PAGE. The location and intensity of the bands can be used to determine if the purification was successful. The concentration of protein produced can be found using UV spectrophotometry.
In this lab, we over-expressed gbr22 in E. coli by cloning and harvested the pure protein. The results were finally confirmed with an SDS-PAGE. We can use the purification of gbr22 as a model for future protein generation in virtual drug screening. As we move from in silico to wet lab testing, we will need to test the activity of the target without other confounding chemicals. This process will allows us to obtain the proteins needed.
Materials and Methods
Protein Expression: The gene gbr22 was cloned and inserted into pGEM-gbr22. E. coli was then transformed by adding 25ul of bacteria to two 14 ml clear, sterile round-bottom tubes and adding 1ul of pGEM-gbr22 to one of the tubes("DNA tube"). After chilling both tubes on ice for 30 minutes, the two tube were heat shocked in a 42 oC water bath and placed back in ice for 2 more minutes. 200 ul of SOC media were added to the two tubes and incubated for 30 minutes at 37 oC at 250 rpm. Afterwards, 50ul of each bacteria tube were placed in two agar AMP plates and stored in a 37 oC incubator for at least 24 hours. Starter cultures were then grown by adding a colony to a LB/amp media and placing inside a 37 oC shaking incubator at 200-350 rpm for 8 hours. 25 ml of the "DNA LB" was then transferred to two 125 ml Erlenmeyer flasks. After 24 hours, the cell were harvested by pouring the bacteria into a 50ml conical tube and centrifuging for 10 minutes at 5000 rpm at 4 oC. The pellet from each centrifuged tube was saved after draining the spent media. 2.5 ml of 1x PBS and lysozyme was added until final concentration was 1mg/ml. The tube was then placed in a -20 oC freezer.
Protein Purification: The E. coli cells were lysed by adding 2 ul of Benzonase to each 50 ml conical. The lysate was then cleared by centrifuging for 20 minutes at 14000 rpm at 4 oC. The liquid supernatant was then extracted and placed in 15 ml conical tubes. The lysate was further filtered with a .22 um syringe filter. The lysate was finally purified using a hexahistone affinity tag and Ni-NTA resin. The purified protein was then run through a UV-Vis spectrophotometer at 280 nm and at maximal wavelength.
Protein Characterization: The purified protein was run through an SDS-PAGE. The MW standard used was PageRuler Prestained Protein Ladder. The gen was run for 25 minutes at 200 V.
Results
The pGEM-gbr22 had 10 colonies growing on it.
The control plate with ampicillin had no growth since the bacteria were not transformed by the plasmid. Therefore, the bacteria were not resistant to the antibiotic. The lack of growth indicates that the techniques used throughout the lab did not involve contamination with the other culture.
The starter cultures had a purple/pink tint, indicating that protein was produced, but had impurities.
The pellets, although filled with cell components became much more purple since they were more concentrated.
The SDS-PAGE gel showed decreasing amounts of protein in each lane 5-10. Sample 1 is missing since the tube could not be found.
Yield Calculations:
280 nm:
A= εlc
.054= 38850 L/(mol cm )*1cm*c
c= .355 mg/ml
.355 mg/ml * 50ml = 17.75 mg
574nm:
A= εlc
10(.067) = 118300 L/(mol cm )*1cm*c
c=.145mg/ml
.145 mg/ml * 50ml = 7.25 mg
The band in sample 5 (purified protein) was lined with the 25 kDa mark. There was a large amount of contaminate from the previous sample, indicating that the initial imidazole flow did not work. The estimated purity was 10% for elution 1. However, elution 2 had a very defined single line, even though there was not supposed to be one. The purity for sample 6 is close to 100%. This suggests that we used the incorrect imidazole solution when performing the elutions.
Discussion
In this "pilot" lab for protein purification, there were many errors that may have contaminated the results. Although there were many major errors, it seems that we were still able to obtain a small amount of reasonably pure gbr22.
One particularly large source of error was when we were performing the elutions. When the lysate was placed inside the column, the tip was not capped and flowed into the waste beaker. We had to reuse this liquid and ran it through the column again, increasing the potential for contaminants. Also, the wrong imidazole concentration was used initially for elution 2. As seen in figure 5B, the elution 1 has many bands even though this is the purified protein and there should be one band. Elution 2, which is supposed to be devoid of any bands, shows one band. This indicates that the procedure was performed incorrectly or that the optimal imidazole concentration was actually 250mM. The spectrophotometer results also indicated a large amount of error. The calculated yields were off by more than a factor of 2. This suggests that there were other contaminants in the sample that contributed to the spectrograph.
Many of these errors could be attributed to clumsiness and inexperience handling the materials (especially adding 5 ladders to the gel). Our results could be improved by simply being more careful and taking more time completely the delicate parts of the procedure.
Conclusion
Despite various errors, we still were able to obtain a pure sample of gbr22 so we can still consider this lab to be successful. We can use this purification technique in the future to produce pure samples of a target protein for protein assay when testing ligands in virtual screening. In addition, purification can be applied on a larger scale to actually produce successful drugs.
References
Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.